Lyophilized Preparation of Botulinum Toxin

ABSTRACT

There are provided a lyophilized preparation of botulinum toxin without a protein stabilizer derived from animals. The lyophilized preparation of botulinum toxin according to the present invention can maintain an activity of botulinum toxin, and also exhibit excellent long-term storage stability even under conditions of high temperature, which may occur when botulinum toxin is stored, delivered, and processed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/533,735, filed Nov. 5, 2014, which is a continuation of U.S. patentapplication Ser. No. 14/008,326, filed Dec. 10, 2013, which is anational stage application under 35 U.S.C. §371 of InternationalApplication PCT/KR2012/002418, filed Mar. 30, 2012, which claimspriority to, and the benefit of, Korean Patent application No.2011-0026577, filed on Mar. 31, 2011, and Korean Patent Application No.2012-0033374, filed on Mar. 30, 2012, the disclosures of each of whichare incorporated herein by reference in their entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to a lyophilized preparation of botulinumtoxin without a protein stabilizer derived from animals.

2. Discussion of Related Art

Botulinum toxin, which is a polypeptide product of Clostridiumbotulinum, anaerobic bacteria, is a toxic material that specificallyaffects a nerve cell. Although botulinum toxin originally is a toxicmaterial causing death, in recent years, it is used for treatingcervical dystonia, blepharospasm, hyperhidrosis, strabismus, achalasia,neurogenic bladder, urologic disease, migraine, and the like. As anexample of use of botulinum toxin as a pharmaceutical composition, thereis Meditoxin Inj. being sold by the present inventors now.

Many proteins having a medicinal effect exhibit a property ofadhesiveness to a solid surface. Therefore, when the proteins areinjected to a container, some of the proteins adhere to an inner wall ofthe container, thereby causing loss of an active component. In addition,since protein may easily be oxidized or degraded into small fragments,it is necessary to add a stabilizer as a material capable of preventingoxidation and degradation of the protein.

Recently, albumin and gelatin are used as a stabilizer for botulinumtoxin. Loss of protein active components may be decreased by reducingprotein denaturation caused due to protein adhesion or dilution whenalbumin is injected into a container. Gelatin is obtained by collagenhydrolysis and sometimes may be used instead of albumin. However, sincealbumin and gelatin are proteins derived from animals, there is a dangerof pathogens derived from blood or latent infection. Therefore, astabilizer which is not derived from animals and also does not causeactivity loss of botulinum toxin is needed.

In this regard, the present inventors disclosed a pharmaceutical liquidcomposition of botulinum toxin including botulinum toxin, methionine,and polysorbate 20 that exhibits long-term stability at normaltemperature in Korean Patent Publication No. 2009-0005963. However, insuch a liquid composition, it is difficult to maintain stability ofbotulinum toxin at a high temperature higher than normal temperature.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a lyophilizedpreparation of botulinum toxin, in which storage stability can bemaintained for a long period of time at a high temperature higher thannormal temperature.

For the conventional preparation of botulinum toxin, stability ofbotulinum toxin can be maintained at a refrigerant temperature or normaltemperature. However, it is difficult to maintain an activity ofbotulinum toxin for a long period of time at a high temperature.Therefore, the present inventors developed a lyophilized preparation ofbotulinum toxin having excellent storage stability, in which an activityof botulinum toxin can be maintained for a long period of time evenacross a wide temperature range, for example, a freezing temperature, arefrigerant temperature, normal temperature, and a high temperature.

Therefore, the present invention provides a pharmaceutical lyophilizedpreparation comprising 1) botulinum toxin; 2) polysorbate; and 3)methionine; and one or more components selected from the groupconsisting of 4) sugar, sugar alcohol, and an ionic compound.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the present invention will be described in detail withreference to examples and comparative examples. However, the presentinvention is not limited to these examples.

The lyophilized preparation of botulinum toxin according to the presentinvention comprises 1) botulinum toxin, 2) polysorbate, and 3)methionine, which have been added to the conventional liquidpreparation; and further includes one or more selected from the groupconsisting of 4) sugar, sugar alcohol, and an ionic compound as anadditional component.

The additional component functions as maintaining an activity ofbotulinum toxin, and also stabilizing the activity even at a hightemperature higher than normal temperature when bolulinum toxin isprepared in a form of a lyophilized formulation. For a compositionincluding 1) botulinum toxin; 2) polysorbate; and 3) methionine, itsstability decreases when being lyophilized, and also decreases at a hightemperature higher than normal temperature even when prepared in aliquid preparation. However, the lyophilized preparation of botulinumtoxin according to the present invention can maintain the activity ofbotulinum toxin even at a high temperature higher than normaltemperature, and also can have excellent long-term storage stability.

The botulinum toxin which is included in the lyophilized preparationaccording to the present invention may be derived from Clostridiumbotulinum. The botulinum toxin which is included in the lyophilizedpreparation according to the present invention may be isolated andpurified from those strains through known methods, or commerciallyavailable products may be used as the botulinum toxin.

The botulinum toxin which is included in the lyophilized preparationaccording to the present invention may be any selected from the groupconsisting of Botulinum Serotypes A, B, C, D, E, F, and G. The botulinumtoxin is divided into Serotypes A, B, C, D, E, F, and G according to animmunological distinguishing method. It is known that the botulinumtoxins of all Serotypes inhibit a secretion of acetylcholine, which is asignaling molecule in a neuromuscular junction, thereby generating aneffect of neural paralysis, and different Serotypes may affect differentanimal species and have different degrees of paralysis, durations, andthe like.

Meanwhile, when a toxin protein is produced by Clostridium botulinum,the botulinum toxin protein is produced by forming various complexeswith various hemagglutinine proteins and non-hemagglutinine proteins,which assist and protect a function of botulinum toxin protein. Thebotulinum toxin which is included in the lyophilized preparationaccording to the present invention may include a complex form with acomplexing protein and a form without a complexing protein. The activityof botulinum toxin is unaffected by whether or not the complexingprotein is included.

In the lyophilized preparation of botulinum toxin according to thepresent invention, polysorbate, which is one of stabilizers of botulinumtoxin, is a nonionic surfactant and mainly used as an emulsifying agentin the field of pharmaceuticals or food. A type of polysorbate includespolysorbates 20, 40, 60, 80, and 100 on the basis of the total number ofan oxyethylene group. For the lyophilized preparation of botulinum toxinaccording to the present invention, all of those polysorbates can beused. The polysorbate may be included in an amount of 0.01 to 2 mg withrespect to 100 units of botulinum toxin. Within the above range, anactivity of botulinum toxin can be maintained even at a high temperaturehigher than normal temperature, and also storage stability can bemaintained for a long period of time.

In addition, methionine, a stabilizer, is used instead of an animalprotein such as albumin and gelatin as a stabilizer of botulinum toxin.Methionine may be included in an amount of 0.01 to 10 mg with respect to100 units of botulinum toxin. Within the above range, an activity ofbotulinum toxin can be maintained even at a high temperature higher thannormal temperature, and also storage stability can be maintained for along period of time.

The lyophilized preparation of botulinum toxin according to the presentinvention further includes at least one of 4) sugar, sugar alcohol, oran ionic compound as an additional component in addition to methionineand polysorbate, unlike the conventional liquid preparation.

Sugar is known to prevent denaturation of macromolecules. An example ofsugar that may be used for the lyophilized preparation according to thepresent invention includes, but is not limited to, trehalose, sucrose,maltose, fructose, raffinose, lactose, glucose, or the like. Such sugarmay be included in an amount of 0.1 to 50 mg with respect to 100 unitsof botulinum toxin. Within the above range, an activity of botulinumtoxin can be maintained even at a high temperature higher than normaltemperature, and also storage stability can be maintained for a longperiod of time.

Sugar alcohol is known to stabilize macromolecules when freeze-dried orin a liquid state, and to prevent denaturation. An example of sugaralcohol that may be used for the lyophilized preparation according tothe present invention includes, but is not limited to, cyclodextrin,mannitol, sorbitol, glycerol, xylitol, inositol, or the like. The sugaralcohol may be included in an amount of 0.1 to 50 mg with respect to 100units of botulinum toxin. Within the above range, an activity ofbotulinum toxin can be maintained even at a high temperature higher thannormal temperature, and also storage stability can be maintained for along period of time.

In addition, an ionic compound means salt or a buffer. An ionic compoundreacts with macromolecules through specific or non-specific binding. Thesalt may increase thermostability and solubility, and may decrease adegree of aggregation. However, it is important to note that a proteinmay tend to be denatured at a high concentration of salt. An example ofthe ionic compound includes, but is not limited to, sodium chloride,sodium phosphate, ammonium phosphate, magnesium sulfate, sodium acetate,sodium lactate, sodium succinate, sodium propionate, potassiumphosphate, or the like. The ionic compound may be included in an amountof 0.1 to 10 mg with respect to 100 units of botulinum toxin. Within theabove range, an activity of botulinum toxin can be maintained even at ahigh temperature higher than normal temperature, and also storagestability can be maintained for a long period of time.

The lyophilized preparation of botulinum toxin according to the presentinvention is prepared from a culture of Clostridium botulinum culturedin a specific medium, but the present invention is not limited thereto.A complex of botulinum toxin is purified from the culture solutionthrough a series of acid precipitations to obtain a crystal complex ofbotulinum toxin composed of an active high-molecular weight toxinprotein and a relevant haemagglutinin protein. The crystal complex isdissolved in a solution including salt water and a stabilizer, and thenfreeze-dried to produce the lyophilized preparation of botulinum toxin.

The lyophilized preparation of botulinum toxin according to the presentinvention can maintain an activity of botulinum toxin, and also exhibitexcellent long-term storage stability even under conditions of hightemperature, which may occur when botulinum toxin is stored, delivered,and processed.

The present invention can be used as a medicine for treating cervicaldystonia, blepharospasm, hyperhidrosis, strabismus, achalasia,neurogenic bladder, urologic disease, migraine, and the like.

The advantages and characteristics of the present invention, and methodsfor obtaining the advantages and characteristics of the presentinvention, will be apparent with reference to the exemplary embodimentsdescribed in detail below. However, the present invention is not limitedto any aspect of the exemplary embodiments disclosed below and may beimplemented in various different forms. The exemplary embodiments areonly provided to enable those skilled in the art to embody and practicethe present invention. The technical spirit and scope of the presentinvention is defined by the appended claims.

<Example 1>Production of Lyophilized preparation of Botulinum Toxin

A lyophilized preparation of botulinum toxin according to the presentinvention was prepared by lyophilizing (or freeze-drying)g a sterilizedpreparation solution including botulinum toxin, methionine, andpolysorbate, and sugar or sugar alcohol and/or an ionic compound.

(Botulinum Toxin Stability Test)

Stability of botulinum toxin was determined by confirming continuity ofactivity after storing for a certain period of time and the continuityof activity of botulinum toxin was measured by checking a lethality ofmice or mouse LD₅₀. A dosage form of the lyophilized preparation wasstored at 40° C. and a relative humidity of 70% for 30 days and thendissolved in physiological saline. Then, the botulinum toxincorresponding 2.5 LD₅₀ IU was abdominally injected to three mice. Whentwo or more mice died, it was determined that stability continued, whichis expressed as mortality in the following Table. When the micemortality is 50% or more, it may be estimated that activity of botulinumtoxin is maintained.

(Titrimetry)

A titrimetry was performed as follows. 2.8 mL of physiological salinewas added to two vials including specimens, respectively. 4.4 mL of thespecimen was taken from the vial, and then 1.45 mL of physiologicalsaline was added to the specimen to obtain a Test solution 1. 1.45 mL ofphysiological saline was added to 4.4 mL of Test solution 1 to obtain aTest solution 2. By the same method, dilution of the solution wasrepeatedly performed eight times to obtain each of the test solutions.For Test solutions 3 to 6, 0.1 mL of each of the test solutions wasabdominally injected to 10 mice (CD1, female) having a weight of 17 to22 g, and then after 3 days, a lethality was measured. The results werestatistically processed by using a Probit method to obtain mouse LD₅₀and titer.

<Example 2> Selection of Stabilizer of Botulinum Toxin

(1) Selection of combination of methionine and polysorbate

In the combination of human serum albumin and polysorbate, which arecomponents of a conventionally known stabilizer of botulinum toxin, astabilizer for the exchange of the human serum albumin was selected.

TABLE 1 Mortality Composition of liquid formulation after Botulinumstoring Polysorbate 20 toxin Stabilizer for 30 (mg/mL) (unit/mL)(Concentration) days (%) 2 100 — 0 HSA (5 mg/mL) 100 L-methionine (20mM) 100 L-arginine (50 mM) 0 Histidine (10 mM) 0 Mannitol (50 mg/mL) 0Sorbitol (50 mg/mL) 0 Sucrose (50 mg/mL) 0 Lactose (50 mg/mL) 0

From the above results, it was estimated that the combination of HSA andpolysorbate 20 could be replaced with the combination of methionine andpolysorbate 20 as a stabilizer.

Next, for the combination of methionine and polysorbate 20 selected as astabilizer of botulinum toxin, stability tests of botulinum toxin wereperformed according to various concentration changes of methionine andpolysorbate 20.

Table 2 shows stability test results (mortality (%)) of botulinum toxinunder conditions of various concentrations of methionine and polysorbate20 in the case of storing for 30 days, and Table 3 shows stability testresults (mortality (%)) of botulinum toxin under conditions of variousconcentrations of methionine and polysorbate 20 in the case of storingfor 60 days. The concentration of botulinum toxin in the botulinum toxinliquid composition of the above test was 100 units/mL.

TABLE 2 Methionine (mM) Concentration 1 5 10 25 50 75 100 Polysorbate0.1 100 100 80 100 100 100 100 20 (mg/mL) 0.5 100 100 80 100 100 100 1002.5 100 100 100 100 100 100 100 10 100 100 100 100 100 100 100 20 100100 100 100 100 100 100 25 80 100 100 80 80 100 100

TABLE 3 Methionine (mM) Concentration 1 5 10 25 50 75 100 Polysorbate0.1 100 100 80 100 100  80 100 20 (mg/mL) 0.5 100 100 80 100 100 100 1002.5 100 100 100 80 100 100 100 10 0 40 0 100 100 100 40 20 0 80 80 100 —100 60 25 0 0 0 0  80 — 0

As a result of performing a statistical analysis using the resultslisted in Tables 2 and 3, it was assumed that the combination of 25 to75 mM of methionine and 0.1 to 2.5 mg/mL of polysorbate 20 maximallystabilized the botulinum toxin.

(2) Selection of Additional Component

When the methionine and polysorbate 20 selected from Tables 2 and 3 wereused as a lyophilized preparation, the contents of methionine andpolysorbate 20 were calculated to be in the ranges of 0.01 to 10 mg and0.01 to 1 mg, respectively. However, when the stabilizer having such acombination was used as the lyophilized preparation, the stability wasnot maintained after storing for 30 days. Therefore, an additionalstabilizer capable of maintaining the stability was selected, as listedin Table 4. At this time, 100 units of botulinum toxin, 3 mg ofmethionine, and 2 mg of polysorbate 20 were used.

TABLE 4 Composition of lyophilized preparation Mortality Additionalstabilizer after storing Sodium Sodium for 30 days chloride phosphateSucrose Mannitol Sorbitol (%) Botulinum toxin + — — — — — 0 Methionine +0.9 mg — — — — 100 Polysorbate 20 — 10 mM — — — 100 — — 0.3 mg  — — 100— — — 40 mg — 100 0.9 mg — — 40 mg — 100 — 10 mM — 40 mg — 100 — 10 mM50 mg — — 100 0.9 mg — 50 mg — — 100 — 10 mM 50 mg — 40 mg 100

As shown in Table 4, it could be confirmed that when the lyophilizedpreparation including only methionine and polysorbate as a stabilizerwas used, the stabilization effect of botulinum toxin was notmaintained, but when at least one of sugar, sugar alcohol, and an ioniccompound was further added in addition to methionine and polysorbate,the stabilization effect was maintained.

Next, a proper content and a type of sugar, sugar alcohol, or an ioniccompound which is further added to the combination of methionine andpolysorbate were tested. At this time, 100 units of botulinum toxin, 2mg of methionine, and 0.2 mg of polysorbate 20 were used.

TABLE 5 Mortality after storing Composition of lyophilized preparationfor 30 Additional stabilizer Content days (%) Botulinum Sucrose 0.3 mg100 toxin + 2.0 mg 100 Methio- 4.0 mg 100 nine + 50 mg 100 Polysor-Trehalose 0.3 mg 100 bate 20 2.0 mg 100 Sorbitol 40 mg 100 Mannitol 40mg 100 Sodium chloride 0.06 mg 0 0.1 mg 100 0.3 mg 100 0.6 mg 100 0.9 mg100 1.2 mg 100 10 mg 100 Sodium sodium hydrogen 0.05 mg 100 phosphatephosphate, anhydrous Sodium dihydrogen 0.101 mg phosphate dihydrate

As shown in Table 5, it could be confirmed that when 0.1 to 50 mg ofsucrose and 0.1 to 10 mg of sodium chloride were added to thecombination of methionine and polysorbate, the stabilization effect wasmaintained. When the content of the additional stabilizer was the aboverange or less, there was no stabilization effect, while when the contentof the additional stabilizer was the above range or more, the stabletype as the lyophilized preparation was not obtained.

Next, the long-time stability test of the lyophilized preparation at ahigh temperature was performed by using titrimetry. At this time, whenthe combination of botulinum toxin + methionine + polysorbate 20 +sodium phosphate + sucrose was used as the lyophilized preparation, 100units of botulinum toxin, 0.8 mg of methionine, 0.02 mg of polysorbate20, sodium phosphate (0.05 mg of sodium hydrogen phosphate, anhydrous+0.101 mg of sodium dihydrogen phosphate dehydrate), and 4 mg of sucrosewere used. When the combination of botulinum toxin + methionine +polysorbate 20 + sodium chloride + sucrose was used as the lyophilizedpreparation, 100 units of botulinum toxin, 0.2 mg of methionine, 0.02 mgof polysorbate 20, 2 mg of sodium chloride, and 4 mg of sucrose wereused. When the lyophilized preparation including human serum albumin wasused as a control group, 0.5 mg of human serum albumin and 0.9 mg ofsodium chloride were used.

TABLE 6 Titer (units) 2 4 8 12 24 Composition of lyophilized 0 weekweeks weeks weeks weeks weeks preparation titer titer titer titer titertiter Botulinum toxin + —  0  0 — — — Human serum albumin + Sodiumchloride Botulinum toxin + 109 100  80 100 95 103 Methionine + (120)(95) (95) (120) Polysorbate 20 + Sodium phosphate (Sodium chloride) +Sucrose

As shown in Table 6, it could be confirmed that when the combination ofbotulinum toxin, methionine, polysorbate, sodium phosphate or sodiumchloride, and sucrose was used as the lyophilized preparation, thestabilization effect was maintained for about 6 months.

While the invention has been shown and described with reference tocertain exemplary embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims.

What is claimed is:
 1. A pharmaceutical lyophilized preparationcomprising: botulinum toxin, polysorbate, and methionine; and one ormore components selected from the group consisting of sugar, sugaralcohol, and an ionic compound, wherein the preparation is free ofalbumin, and wherein stability of the botulinum toxin is maintained forat least 30 days at room temperature.
 2. The pharmaceutical lyophilizedpreparation of claim 1, wherein the botulinum toxin is selected from thegroup consisting of botulinum serotypes A, B, C, D, E, F, and G.
 3. Thepharmaceutical lyophilized preparation of claim 1, wherein the botulinumtoxin is selected from the group consisting of a form without acomplexing protein and a form with a complexing protein.
 4. Thepharmaceutical lyophilized preparation of claim 1, wherein thepolysorbate is any one of polysorbates 20, 40, 60, 80, and
 100. 5. Thepharmaceutical lyophilized preparation of claim 1, wherein thepolysorbate is included in an amount of 0.01 to 2 mg with respect to 100units of the botulinum toxin.
 6. The pharmaceutical lyophilizedpreparation of claim 1, wherein the methionine is included in an amountof 0.01 to 10 mg with respect to 100 units of the botulinum toxin. 7.The pharmaceutical lyophilized preparation of claim 1, wherein the sugaris one or more selected from the group consisting of trehalose, sucrose,maltose, fructose, raffinose, lactose, and glucose.
 8. Thepharmaceutical lyophilized preparation of claim 1, wherein the sugar isincluded in an amount of 0.1 to 50 mg with respect to 100 units of thebotulinum toxin.
 9. The pharmaceutical lyophilized preparation of claim1, wherein the sugar alcohol is one or more selected from the groupconsisting of cyclodextrin, mannitol, sorbitol, glycerol, xylitol, andinositol.
 10. The pharmaceutical lyophilized preparation of claim 1,wherein the sugar alcohol is included in an amount of 0.1 to 50 mg withrespect to 100 units of the botulinum toxin.
 11. The pharmaceuticallyophilized preparation of claim 1, wherein the ionic compound is one ormore selected from the group consisting of sodium chloride, sodiumphosphate, ammonium phosphate, magnesium sulfate, sodium acetate, sodiumlactate, sodium succinate, sodium propionate, and potassium phosphate.12. The pharmaceutical lyophilized preparation of claim 1, wherein theionic compound is included in an amount of 0.1 to 10 mg with respect to100 units of the botulinum toxin.
 13. A pharmaceutical lyophilizedpreparation comprising: botulinum toxin, polysorbate, methionine, sugar,and an ionic compound, wherein the preparation is free of albumin, andwherein stability of the botulinum toxin is maintained for at least 30days at room temperature.
 14. A pharmaceutical lyophilized preparationcomprising: botulinum toxin, polysorbate 20, methionine, sucrose, andsodium chloride, wherein the preparation is free of albumin, and whereinstability of the botulinum toxin is maintained for at least 30 days atroom temperature.